DNA nanoparticle formulations for optimal ocular gene delivery

用于最佳眼部基因传递的 DNA 纳米颗粒配方

• 批准号: 8734431
• 负责人: Muna I. Naash
• 金额: $ 40.03万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2016-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734431
• 关键词: Accounting; Affect; Animals; Automobile Driving; Biodistribution; Biologic Characteristic; Biomedical Engineering; Blindness; Brain; Bypass; Caliber; Cell Nucleus; Cell membrane; Cells; Characteristics; Chemicals; Chemistry; Clinic; Clinical; Cone; Coupled; DNA; Data; Development; Diabetic Retinopathy; Disease; Dose; Drug Formulations; Elements; Engineering; Enhancers; Enzyme-Linked Immunosorbent Assay; Epithelial; Evaluation; Excipients; Exhibits; Eye; Face; Fluorescence; Foundations; Future; GRB10 gene; Gene Delivery; Gene Expression; Gene Transfer; Generations; Genes; Goals; Human; Injection of therapeutic agent; Interphase Cell; Ions; Length; Longevity; Luciferases; Lung; Lysine; Matrix Attachment Regions; Mediating; Methodology; Methods; Mitotic; Modeling; Molecular; Mus; Nanotechnology; Papio; Parkinson Disease; Pathway interactions; Penetration; Peptides; Performance; Photoreceptors; Physics; Plasmids; Polyethylene Glycols; Polylysine; Primates; Procedures; Proteins; Research; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinal Ganglion Cells; Retinal Pigments; Retinitis Pigmentosa; Retinol Binding Proteins; Rodent; Route; Safety; Shapes; Solvents; Structure of retinal pigment epithelium; System; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Toxicology; Transfection; Treatment Efficacy; Vertebral column; Viral Vector; Visual Pathways; Vitelliform macular dystrophy; clinical application; cystic fibrosis patients; density; design; effective therapy; gene delivery system; gene therapy; improved; intravitreal injection; mRNA Expression; macula; macular dystrophy; minimally invasive; mouse model; nanoparticle; non-viral gene therapy; nonhuman primate; novel; particle; polycation; postnatal; preclinical study; prevent; programs; promoter; protein expression; ranpirnase; response; retinal rods; single molecule; therapeutic gene; trafficking; transgene expression; uptake; vector; vision science

DESCRIPTION (provided by applicant): The goal of this program is to advance current DNA nanoparticle (NP) delivery and expression technologies to develop safe and effective therapies for important ocular disorders affecting the photoreceptor (PR) and retinal pigment epithelial (RPE) cells. The program will merge experts with molecular bioengineering, vision science, physics, and chemistry to accelerate essential steps for the generation of effective ocular non-viral gene therapy. The DNA NPs consist of single molecules of DNA compacted with lysine-PEG polycations and have a minimum diameter of 8-11 nm. Their small size, coupled with a specific uptake mechanism that efficiently traffics the NPs to the nucleus and bypasses the lysosomal degradation system, likely accounts for the ability of the NPs to robustly transfect post-mitotic, differentiated cells. The counter-ion used at the time of formulation determines the NP shape and both rod-like and ellipsoidal NPs show robust transfection of ocular cells, including RPE, PRs and retinal ganglion cells (RGCs). Moreover, murine RDS NPs have demonstrated partial phenotypic correction in a retinitis pigmentosa mouse model of RDS haploinsufficiency. Having previously shown proof-of-principle for the effective use of these NPs in the eye, in this application we will take the necessary steps to optimize the particles for clinical ocular use. First, we will optimize the NP formulation (NP shape, size, and chemical composition) for PR- and RPE-specific gene transfer (Aim #1). Second, we will engineer clinically-appropriate DNA vectors (Aim #2) to generate persistent, high levels of transgene expression. Thirds, we will test the ability of these NPs to target the macula in a non-human primate model (baboon) (Aim #3) including an assessment of whether non-invasive intravitreal delivery of NPs can transfect macular/foveal cones. This is a novel and critical development for clinical application of this technology, as many retinal degenerations target the macula. Furthermore, it is a step that cannot be modeled by a mouse system due to the absence of a macula in the rodent retina. We will also conduct toxicology and DNA biodistribution studies in baboons, including a detailed evaluation of brain visual pathways. Building on the documented ability of the current NP formulation to penetrate deep retinal layers, we hypothesize that this efficiency can be improved by the NP formulation optimization program detailed in Aim #1, enabling robust foveal cone gene transfer. In summary, results from this application will facilitate preclinical trial evaluations of DNA NPs for ocular gene delivery.

描述（由申请人提供）：该程序的目的是推进当前的DNA纳米颗粒（NP）传递和表达技术，以开发影响感光体（PR）和视网膜色素上皮（RPE）细胞的重要眼部疾病的安全有效疗法。 该计划将将专家与分子生物工程，视觉科学，物理和化学合并，以加快生成有效的眼科非病毒基因治疗的基本步骤。 DNA NP由用赖氨酸-PEG多阳离子压实的DNA的单分子组成，最小直径为8-11 nm。 它们的尺寸很小，再加上特定的摄取机制，该机制有效地将NPS运输到细胞核并绕过溶酶体降解系统，可能是NPS可靠地转染后有丝质后分化细胞的能力。 配方时使用的反离子决定了 NP形状以及杆状和椭圆形NP都表现出可靠的眼细胞转染，包括RPE，PRS和视网膜神经节细胞（RGC）。 此外，鼠RDS NP已显示出RDS单倍​​不足的视网膜炎小鼠模型中的部分表型校正。 先前显示了有效使用这些NP的原则证明，在此应用中，我们将采取必要的步骤来优化临床眼的颗粒。 首先，我们将优化用于PR和RPE特异性基因转移的NP公式（NP形状，大小和化学组成）（AIM＃1）。 其次，我们将设计适合临床的DNA矢量（AIM＃2），以产生持久的高水平转基因表达。 三分之一，我们将测试这些NP在非人类灵长类动物模型（AIM＃3）中靶向黄斑的能力，包括评估NP的非侵入性玻璃体内递送是否可以转染黄斑/凹锥。 这是该技术临床应用的新颖而重要的发展，因为许多视网膜变性针对黄斑。 此外，由于啮齿动物视网膜中没有黄斑，这是一个步骤，无法通过小鼠系统对其进行建模。 我们还将在狒狒中进行毒理学和DNA生物分布研究，包括对脑视觉途径的详细评估。 基于当前NP公式渗透深视网膜层的证明能力，我们假设可以通过在AIM＃1中详细介绍的NP公式优化程序来提高这种效率，从而实现了强大的foveal锥基因转移。 总而言之，此应用的结果将促进眼科基因递送的DNA NP的临床前试验评估。